Oncostatin M is a 28 KDa glycoprotein that belongs to the interleukin 6 (IL-6) family of cytokines which includes IL-6, Leukaemia Inhibitory Factor (LIF), ciliary neurotrophic factor (CNTF), cardiotropin-1 (CT-1) and cardiotrophin-1 like cytokine (See Kishimoto T et al (1995) Blood 86: 1243-1254), which share the gp130 transmembrane signalling receptor (See Taga T and Kishimoto T (1997) Annu. Rev. Immunol. 15: 797-819). OSM was originally discovered by its ability to inhibit the growth of the melanoma cell line A375 (See Malik N (1989) et al Mol Cell Biol 9: 2847-2853). Subsequently, more effects were discovered and it was found to be a multifunctional mediator like other members of the IL-6 family. OSM is produced in a variety of cell types including macrophages, activated T cells (See Zarling J M (1986) PNAS (USA) 83: 9739-9743), polymorphonuclear neutrophils (See Grenier A et al (1999) Blood 93:1413-1421), eosinophils (See Tamura S et al (2002) Dev. Dyn. 225: 327-31), dendritic cells (See Suda T et al (2002) Cytokine 17:335-340). It pancreas, kidney, testes, spleen stomach and brain (See Znoyko I et al (2005) Anat Rec A Discov Mol Cell Evol Biol 283: 182-186), and bone marrow (See Psenak O et al (2003) Acta Haematol 109: 68-75) Its principle biological effects include activation of endothelium (See Brown T J et al (1993) Blood 82: 33-7), activation of the acute phase response (See Benigni F et al (1996) Blood 87: 1851-1854), induction of cellular proliferation or differentiation, modulation of inflammatory mediator release and haematopoesis (See Tanaka M et al (2003) 102: 3154-3162), re-modelling of bone (See de Hooge A S K (2002) Am J Pathol 160: 1733-1743) and, promotion of angiogenesis (See Vasse M et al (1999) Arterioscler Thromb Vasc Biol 19:1835-1842) and wound healing.
Receptors for OSM (OSM receptor β, “OSMRβ”) are expressed on a wide range of cells including epithelial cells, chondrocytes, fibroblasts (See Langdon C et al (2003) J Immunol 170: 548-555), neuronal smooth muscle, lymph node, bone, heart, small intestine, lung and kidney (See Tamura S et al (2002) Mech Dev 115: 127-131) and endothelial cells. Several lines of evidence suggest that endothelial cells are a primary target for OSM. These cells express 10 to 20 fold higher numbers of both high and low affinity receptors and exhibit profound and prolonged alterations in phenotype following stimulation with OSM (See Modur V et al (1997) J Clin Invest 100: 158-168). In addition, OSM is a major autocrine growth factor for Kaposi's sarcoma cells, which are thought to be of endothelial origin (See Murakami-Mori K et al (1995) J Clin Invest 96:1319-1327).
In common with other IL-6 family cytokines, OSM binds to the transmembrane signal transducing glycoprotein gp130. A key feature of the gp130 cytokines is the formation of oligomeric receptor complexes that comprise gp130 and one or more co-receptors depending on the ligand (Reviewed in Heinrich P C et al (2003) Biochem J. 374: 1-20). As a result, these cytokines can mediate both the shared and unique biological activities in vitro and in vivo depending on the composition of the receptor complex formed. Human OSM (hOSM) differs from the other IL-6 cytokines in that it can form complexes with gp130 and either one of the two co-receptors, LIFR or the oncostatin receptor (OSMR). FIG. 1 illustrates the interaction between hOSM and gp130, LIFR and OSMR. The crystal structure of hOSM has been solved and shown to comprise a four α helical bundle with two potential glycosylation sites. Two separate ligand binding sites have been identified by site-directed mutagenesis on the hOSM molecule (See Deller M C et al (2000) Structural Fold Des. 8:863-874). The first, called Site II (sometimes “site 2”) interacts with gp130 and the second site, called Site III (sometimes “site 3”), at the opposite end of the molecule interacts with either LIFR or OSMR. Mutagenesis experiments have shown that the binding sites for LIFR and OSMR are almost identical but that a single amino acid mutation can discriminate between the two.
OSM is synthesised as a proprotein containing a hydrophobic 25 amino acid (AA) N terminal signal sequence and a C-terminal propeptide of 33 AA, both of which are cleaved to generate mature OSM. The OSM proprotein does have biological activity but this is significantly increased by cleavage of the C terminal propeptide (see Bruce A. G. et al (1992) Prog. Growth Factor Res. 4: 157-170, Malik N et al (1989) Mol. Cell Biol. 9: 2847-2853). OSM has been described as a “compact, barrel-shaped molecule” with dimensions of approximately 20 {acute over (Å)}×27 {acute over (Å)}×56 {acute over (Å)}. There are four alpha helical regions (helix A 10-37AA, helix B 67-90AA, helix C 105-131AA and helix D 159-185AA, numbering of AA starts after removal of the signal sequence). Helices A and C contain “kinks”. The helices are joined by two overhand loops (AB loop 38-66M, CD loop 130-158 AA) and are arranged as two anti-parallel pairs (A-D and B-C). (See Deller M. C et al (2000) Structure 8; 863-874).
It appears that OSM binding via Site II to gp130 allows binding of another OSM molecule to gp130 by a Site III interaction. OSM will also bind to either LIFR or OSMR via Site II. Thus OSM forms a complex with its receptor consisting of; one gp130, one LIFR or OSMR, and two OSM molecules. (See Sporeno E (1994) J. Biol. Chem. 269: 10991-10995, Staunton D et al (1998) Prot. Engineer 11:1093-1102 and Gearing D. P (1992) Science 225:306-312).
Using mutagenesis, the important residues for Site II OSM-gp130 binding are Gln20, Gly120, Gln16 and Asn124. For Site III OSM-OSMR binding, the important residues are Phe160 and Lys163. The OSM Site II interaction is therefore dependent on Gln20, Gly120, Asn124 and to a lesser extent Gln16 on hOSM. Three complementary residues in gp130 (Phe169, Tyr196 and Glu282) have been identified as of particular note in the interaction between OSM and gp130. (See Deller M et al (2000) Structure 8:863-874, Aasland D et al (2002) J. Mol. Biol 315: 637-646, Timmermann A et al (2000) FEBS Lett. 468: 120-124).
The amino acid sequence starting at position 1 for hOSM is set forth as SEQ. I.D. NO: 13
(SEQ. I.D. NO:13)MGVLLTQRTLLSLVLALLFPSMASMAAIGSCSKEYRVLLGQLQKQTDLMQ DTSRLLDPYIRIQGLDVPKLREHCRERPGAFPSEETLRGLGRRGFLQTLN ATLGCVLHRLADLEQRLPKAQDLERSGLNIEDLEKLQMARPNILGLRNNI YCMAQLLDNSDTAEPTKAGRGASQPPTPTPASDAFQRKLEGCRFLHGYHR FMHSVGRVFSKWGESPNRSRRHSPHQALRKGVRRTRPSRKGKRLMTRGQL PR..
Site II residues of particular note are highlighted in bold and underlined
A cDNA encoding hOSM is set forth in SEQ. I.D. NO:14.
(SEQ. I.D. NO:14)ATGGGGGTACTGCTCACACAGAGGACGCTGCTCAGTCTGGTCCTTGCACT CCTGTTTCCAAGCATGGCGAGCATGGCGGCTATAGGCAGCTGCTCGAAAG AGTACCGCGTGCTCCTTGGCCAGCTCCAGAAGCAGACAGATCTCATGCAG GACACCAGCAGACTCCTGGACCCCTATATACGTATCCAAGGCCTGGATGT TCCTAAACTGAGAGAGCACTGCAGGGAGCGCCCCGGGGCCTTCCCCAGTG AGGAGACCCTGAGGGGGCTGGGCAGGCGGGGCTTCCTGCAGACCCTCAAT GCCACACTGGGCTGCGTCCTGCACAGACTGGCCGACTTAGAGCAGCGCCT CCCCAAGGCCCAGGATTTGGAGAGGTCTGGGCTGAACATCGAGGACTTGG AGAAGCTGCAGATGGCGAGGCCGAACATCCTCGGGCTCAGGAACAACATC TACTGCATGGCCCAGCTGCTGGACAACTCAGACACGGCTGAGCCCACGAA GGCTGGCCGGGGGGCCTCTCAGCCGCCCACCCCCACCCCTGCCTCGGATG CTTTTCAGCGCAAGCTGGAGGGCTGCAGGTTCCTGCATGGCTACCATCGC TTCATGCACTCAGTGGGGCGGGTCTTCAGCAAGTGGGGGGAGAGCCCGAA CCGGAGCCGGAGACACAGCCCCCACCAGGCCCTGAGGAAGGGGGTGCGCA GGACCAGACCCTCCAGGAAAGGCAAGAGACTCATGACCAGGGGACAGCTG CCCCGGTAG
Rheumatoid arthritis (RA) comprises a syndrome of distinct but inter-connected pathogenic processes. These are: local and systemic inflammation, proliferation of synovial cells, angiogenesis and matrix deposition leading to formation of pannus tissue which invades and destroys cartilage and bone, resulting in deformity and disability. Underpinning this pathology is the chronic release of cytokines and inflammatory mediators from cells that enter and take up residence in the inflamed joint and from endogenous joint tissue cells (See Firestein G (2003) in Rheumatology. Eds Hochberg, Silman, Smolen, Weinblatt and Weisman. Pub. Mosby. 855-884). The initiating events in RA are unknown but a wealth of evidence suggests that they involve activation of T lymphocytes by either a foreign or autologous “self” antigen (See Firestein G (2004) J Clin Invest 114: 471-4). The extent to which T cells are required to maintain the ongoing disease processes once they have been initiated is also uncertain although therapeutic agents such as CTLA4lg, which specifically target T cells can be effective in advanced disease (See Kremer J M et al (2003) New Engl J Med 349: 1907-15, Moreland L et al (2004) Annual meeting of the American College of Rheumatology Abstract 1475).
The earliest events in the development of rheumatoid synovitis involve recruitment of mononuclear and polymorphonuclear cells to cross the endothelium in capillaries in the synovial-lining layer. While the polymorphs migrate into synovial fluid (SF) the lymphocytes remain close to the capillaries and may subsequently become organised into ectopic lymphoid follicles. This influx of immune cells is followed by proliferation of fibroblast-like synoviocytes (FLS). Unlike their normal counterparts, RA FLS appear to have escaped from the regulatory processes that result in arrest of proliferation and apoptosis leading to their continuing accumulation (See Yamanishi Y et al (2004) Arthritis Res Ther 7: 12-18). Furthermore, the emerging pannus tissue now develops new blood vessels supported by extracellular matrix to allow further expansion. This process involving fibroblast proliferation, matrix-remodelling and angiogenesis closely resembles an uncontrolled wound-healing event. Monocytes migrate into the developing pannus tissue and undergo differentation into macrophages with a chronically activated phenotype. Similarly B cells undergo terminal differentiation to form long-lived plasma cells which secrete antibodies including rheumatoid factors. The ability of the inflamed synovium to sustain local differentation of myeloid and lymphoid cells is based, in part, on local production of growth factors such as GMCSF and IL-6. Both the FLS and resident mononuclear leukocytes release soluble factors that stimulate further recruitment of inflammatory cells from the blood and, critically, drive the next step in the disease process—the destruction of articular cartilage and re-modelling of bone. Pannus tissue is invasive. Its leading edge secretes destructive enzymes such as MMPs and cytokines that alter the phenotype of cells which maintain the structural integrity of cartilage and bone. As a result, proteoglycans are lost and type II collagen is irreversibly cleaved leading to weakening and loss of cartilage. Bone also undergoes a number of profound changes, which include focal erosions, sub-chondral osteoporosis. Ultimately these changes result in the characteristic deformity and subluxation of the joints seen in advanced RA (See Gordon D and Hastings D (2003) in Rheumatology. Eds Hochberg, Silman, Smolen, Weinblatt and Weisman. Pub. Mosby. 765-780).
RA is a systemic disease, probably as a result of the passage of inflammatory mediators from the joint into the blood. This affects many organ systems in the body including skin, eyes, liver, kidneys, brain and the vascular lining, leading to increased morbidity and mortality (See Matteson E L (2003) in Rheumatology. Eds Hochberg, Silman, Smolen, Weinblatt and Weisman. Pub. Mosby. 781-792). Much of the excess mortality is due to cardiovascular disease caused by atherosclerosis since many of the pathogenic processes involved in the development of rheumatoid synovitis are common to the formation of atherosclerotic plaques.
Treatments for RA aim to control pain reduce inflammation and arrest the processes that result in tissue destruction. Traditionally RA has been treated with non-steroidal anti-inflammatory drugs (NSAIDS), low doses of steroids and so-called disease modifying anti-rheumatic drugs (DMARDS). Low levels of efficacy, slow onset, toxicity, poor tolerability and increasing resistance over time plague the use of these treatments which include methotrexate (MTX), sulphasalazine, gold and Leflunomide. More recently, the introduction of biologic drugs such as Enbrel™, Remicide™ and Humira™, which inhibit the cytokine Tumour Necrosis Factor (TNF), have been a significant advance (See Roberts L and McColl G J (2004) Intern Med J 34:687-93).
It is therefore an object of the present invention to provide a therapeutic approach to the treatment of RA and other diseases and disorders, particularly chronic inflammatory diseases and disorders such as osteoarthritis and psoriasis. In particular it is an object of the present invention to provide immunoglobulins, especially antibodies that specifically bind OSM (e.g. hOSM, particularly Site II thereof) and modulate (i.e. inhibit or block) the interaction between OSM and gp130 in the treatment of diseases and disorders responsive to modulation of that interaction.
There is increasing evidence to support the hypothesis that modulating OSM-gp130 interaction maybe of benefit in the treatment of such diseases and disorders.
Clinical Evidence
OSM is found in the SF of human RA patients (See Hui W et al (1997) 56: 184-7). These levels correlate with; the number of neutrophils in SF, levels of TNF alpha (sometimes “TNF”) in SF, and markers of cartilage destruction (Manicourt D H et al (2000) Arthritis Rheum 43: 281-288). Furthermore, the synovial tissue from RA patients secretes OSM spontaneously ex vivo (See Okamoto H et al (1997) Arthritis and Rheumatism 40: 1096-1105). It has also been demonstrated that OSM is present in synovial macrophages (Cawston T E et al (1998) Arthritis Rheum 41: 1760-1771) and as discussed earlier, OSM receptors and gp130 are expressed on endothelial cells, synovial fibroblasts, chonodrocytes and osteoblasts. Furthermore, cells infiltrating atherosclerotic plaques and aortic aneurysms express OSM suggesting an association of this cytokine with chronic inflammation (See Mirshahi F et al (2001) Ann NY Acad Sci 936: 621-4).
In Vitro Evidence
Endothelial cells express ten to twenty times the number of OSM receptors than other cell types (See Brown T J et al (1991) J Immunol 147: 2175-2180, Linsley P S et al (1989) J Biol Chem 264: 4282-4289). OSM alone, or synergistically in combination with other cytokines, activates endothelium to release cytokines and chemokines and bind neutrophils, monocytes and lymphocytes mediating their extravasation into synovial tissue (See Modur V et al (1997) J Clin Invest 100: 158-168). OSM has also been demonstrated to be a potent stimulator of angiogenesis (See Vasse M et al (1999) Aterioscler Thromb Vasc Biol 19: 1835-1842) and activation and proliferation of synovial fibroblast (FLS) cells (thus facilitating the formation of pannus tissue, the release of IL-6, MMPs) and acts synergistically with TNF and IL-1 to induce this mediator release (See Langdon C et al (2000) Am J Pathol 157: 1187-1196). OSM has also been demonstrated to induce (with IL-1) collagen and proteoglycan release from cartilage (See Cawston T et al (1995) Biochem Biophys Res Commun 215: 377-385). Furthermore, OSM induces acute phase protein release and production of IL-6 receptor from hepatocytes (See Cichy J et al (1997) J Immunol 159: 5648-5643, Kurash J K (2004) Exp Cell Res 292: 342-58) and may therefore contribute to the systemic effects of rheumatoid inflammation including fatigue. In addition, OSM induces osteoclast differentiation and activity in vitro (See Palmqvist P et al (2002) J Immunol 169: 3353-3362).
In Vivo Evidence
Adenoviral expression of murine OSM (mOSM) in the joints of normal mice results in a severe inflammatory and erosive arthritis (See Langdon C et al (2000) Am J Pathol 157: 1187-1196). Similarly aggressive disease is seen in knockout mice lacking TNF, IL-1, IL-6 and iNOS following adenoviral mOSM delivery (See de Hooge A S K et al (2003) Arthritis and Rheumatism 48:1750-1761), demonstrating that OSM can mediate all aspects of arthritis pathology. Mouse OSM expression using an adenovirally expressed mOSM vector causes damage to the growth plate typical of Juvenile Idiopathic Arthritis (See de Hooge A S K et al (2003) Arthritis and Rheumatism 48:1750-1761). In an experimental model of collagen induced arthritis, an anti-OSM antibody administered therapeutically to mice prevented all further progression of disease. Similar results were seen when anti-OSM was administered prophylatically to mice with pristane induced arthritis, a relapsing/remitting model reminiscient of the human disease (See Plater-Zyberk C et al (2001) Arthritis and Rheumatism 44: 2697-2702). In monkeys, OSM injected subcutaneously induces an acute phase response and local chronic inflammation (See Loy J K et al (1999) Toxicol Pathol 27: 151-155). OSM has been demonstrated to induce mononuclear and PMN infiltration and proteoglycan release when injected into goat joints (See Bell M C et al (1999) Arthritis Rheum 42: 2543-2551). Transgenic over-expression of mOSM in mouse lymph nodes results in extrathymic T cell maturation, proliferation of memory T cells and failure to deplete autoimmune T cells (See Louis I et al (2003) Blood 102: 1397-1404). Transgenic over-expression of OSM in the pancreas causes extensive fibrosis similar to that seen in advanced RA synovium (See Malik N et al (1995) Mol Cell Biol 15: 2349-2358).
In WO99/48523, we disclose the use of OSM antagonists in the treatment of inflammatory diseases and disorders. This disclosure used an anti-mouse OSM antibody in a murine model of arthritis.
All patent and literature references disclosed within the present specification are expressly and entirely incorporated herein by reference.